The present invention relates to the novel protein termed steroid receptor coactivator-one (xe2x80x9cSRC-1xe2x80x9d), nucleotide sequences encoding SRC-1, as well as various products and methods useful for augmenting or downregulating the activity of one or more steroid receptors.
The following description of the background of the invention is provided to aid in understanding the invention but is not admitted to be prior art to the invention.
Transcription is a fundamental biological process whereby an RNA molecule is formed upon a DNA template by complementary base pairing that is mediated by RNA polymerase II. Accumulated evidence indicates that numerous general transcription factors undergo a defined order of assembly at promoter-DNA elements to assure RNA polymerase II binding and initiation of transcription of target genes (for review see Zawel, L. and Reinberg, D. (1995), Ann. Rev. Biochem. 64, 533-561).
Activation of transcription can be achieved by direct interaction of activators with one or more components of the basal transcriptional machinery. Direct interaction between activators and the basal transcription machinery has been described for several activators (Stringer, K. F. et al., (1990), Nature 345, 783-785; Kashanchi, F. et al., (1994), Nature 367, 295-299; Sauer, F. et al., (1995), Nature 375, 162-164).
Optimal transactivation by an activator is likely to require additional factors termed adaptors or coactivators. These factors seem to play a key regulatory role in bridging or stabilizing the activator with general transcription factors in the core transcriptional machinery. The ability of an activator to squelch or inhibit transactivation of a target gene by another transactivator suggests that they compete for a limited amount of cofactor(s) required for the transactivation process and furthers the concept that coactivators are required for efficient transactivational function (Flanagan, P. M. et al., (1991), Nature 350, 436-438).
It has been postulated that steroid receptors regulate transcription via interactions with the basal transcriptional machinery. However, the finding that squelching occurs between members of the steroid receptor superfamily, indicates that an additional factor(s) or coactivator(s) is important for efficient ligand-inducible target gene expression by members of this superfamily (Meyer, M. E. et al., (1989), Cell 57, 433-442; Conneely, O. M. et al., (1989), xe2x80x9cPromoter specific activating domains of the chicken progesterone receptor.xe2x80x9d In Gene Regulation by Steroid Hormones IV. A. K. Roy and J. Clark, eds. (New York, Berlin, Heidelberg, London, Paris, Tokyo: Springer-Verlag), pp. 220-223; Bocquel, M. T. et al., (1989), Nucleic Acids Res. 17, 2581-2594; Shemshedini, L. et al., (1992), J. Biol. Chem. 261, 1834-1839). No such functional coactivator for this superfamily has previously been identified.
Steroid receptors belong to a superfamily of ligand inducible transcription factors which regulate hormone responsive genes and thereby affect several biological processes including cell growth and differentiation. The steroid/thyroid hormone receptor superfamily can be divided into two types (termed A and B) based on their characteristic association with heat shock proteins, binding to DNA and their ligand-dependent transactivation function (Tsai, M. -J. and O""Malley, B. W. (1994), Ann. Rev. Biochem. 63, 451-486).
One steroid receptor, the human progesterone receptor (hPR), is expressed in cells as two isoforms: PRB of 120 kDa and PRA of 94 kDa. The A isoform is a shorter transcript of PR, lacking the most N-terminal 164 amino acids of the B receptor (Kastner, P. et al., (1990), EMBO J. 9, 1603-1614; Wei, L. L et al., (1987), Biochem. 26, 6262-6272). Although they display similar ligand specificities and DNA-binding affinities in vitro, the transcriptional activity of the two receptor isoforms show different promoter and cell specificities when assayed in intact cells (Chalepakis, G. et al., (1988), Cell 53, 371-382; Tora, L. et al., (1988), Nature 333, 185-188; Tung, L. et al., (1993), Mol. Endocrinol. 7, 1256-1265).
Like other members of the steroid receptor superfamily, the hPRs are modular proteins containing a ligand binding domain (LBD) at the C-terminus and a centrally located DNA binding domain (DBD). Two regions in hPR have been thought to contain transcriptional activation functions (AFs). One is located at the N-terminus (AF1) and the other (AF2) is located within the LBD (Tora, L. et al., (1989), Cell 59, 477-487; Gronemeyer, H. (1991), Ann. Rev. Genet. 25, 89-123). Recent results indicate that the hPRBspecific 164 amino acid fragment may contain an additional activation function (Sartorius, C. A. et al., (1994), Mol. Endocrinol. 8, 1347-1360) that is required for maximal transactivation of the full-length receptor.
Activation of a steroid receptor is a complex multi-step process that involves structural and functional alterations of receptor which promote specific binding to DNA hormone-responsive elements (HREs) to modulate the target gene expression (for review see Tsai, M. -J. and O""Malley, B. W. (1994), Ann. Rev. Biochem. 63, 451-486). Thus, steroid receptors must undergo a rather complex multi-step activation process to achieve their ultimate transactivational function.
Coactivators have been implicated widely in nuclear steroid receptor function. Transcriptional interference experiments between members of the steroid receptor superfamily suggested that coactivators are limiting and interact, either directly or indirectly, with the receptor protein in vivo to modulate transcription. However, as noted above, no such functional coactivator for this superfamily has previously been identified.
The present invention relates to SRC-1 polypeptides, nucleic acids encoding such polypeptides, cells containing such nucleic acids, antibodies to such gene products, assays utilizing such polypeptides, and methods relating to all of the foregoing. In particular, this invention relates to methods for augmenting or downregulating the activity of one or more steroid receptors.
The present invention is based upon the isolation and characterization of a new protein which we have designated steroid receptor coactivator-1, or SRC-1. We have determined that modulation of SRC-1 activity is useful in therapeutic procedures and thus the present invention provides several agents and methods useful for modulating steroid hormone responses and activities, including modulation of the activity of other transactivators.
The isolated, purified, and/or enriched SRC-1 polypeptides and/or nucleic acids can be used to transactivate a steroid receptor and thereby promote the level of transcription in an organisms or cell. Administration of the appropriate material can be accomplished by one skilled in the art using methods described herein. For example, one or more transfected and/or transformed cells can be used to perform a gene therapy based treatment where activity of steroid receptors may be involved. Examples of disorders or conditions that involve the activity of steroid receptors include malignancies of the reproductive endocrine system and inflamation and immunity disorders, such as those described in U.S. patent application Ser. No. 08/479,913, filed Jun. 7, 1995, incorporated herein by reference in its entirety, including any drawings. Examples of other disorders or conditions are listed in references available to those skilled in the art such as the Physicians"" Desk Reference and include endocrine disorders, rheumatic disorders, collagen disorders, dermatologic diseases, allergic states, ophthalmic diseases, gastrointestinal diseases, respitory diseases, hematologic disorders, breast cancer, endometriosis, hyperproliferative disorders including cancer and others. Alternatively, methods of the invention may be used to inhibit transcription. For example, a truncated form of SRC-1 can be used as a dominant negative inhibitor of receptor activity.
We describe herein the cloning and characterization of a cDNA encoding a protein required for hPR transactivational function, hereafter termed steroid receptor coactivator-one (SRC-1). SRC-1 directly and specifically interacts with the ligand binding domain (LBD) of hPR in a hormone-dependent manner. Binding of the antagonist RU486 to the receptor protein abolishes this interaction. Coexpression of SRC-1 with steroid receptors enhances ( greater than 10 fold) the hormone-induced transcription of a cellular target gene without altering the basal activity. Furthermore, overexpression of SRC-1 can reverse the ability of ER to squelch PR-mediated transactivation. Finally, coexpression of a truncated form of SRC-1, which retains the ability to interact with receptor, results in a dominant-negative inhibition of receptor activity. SRC-1 thus encodes a protein that fulfills the properties of a coactivator which ensures efficient ligand-dependent activity of steroid receptors on target genes.
Thus, in a first aspect the invention features an isolated, enriched, or purified nucleic acid encoding a SRC-1 polypeptide.
By xe2x80x9ca SRC-1 polypeptidexe2x80x9d is meant 25 (preferably 30, more preferably 35, most preferably 40) or more contiguous amino acids set forth in the full length amino acid sequence of FIGS. 1A-1E, or a functional derivative thereof as described herein. In certain aspects, polypeptides of 50, 100, 425, 430, 435, 440 or more amino acids are preferred. The SRC-1 polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained. Such functional activity can be, for example, (1) the ability to interact with the PR in an agonist specific manner, (2) the ability to enhance the hormone-induced transcriptional activity without altering basal activity of the promoter, (3) the ability to stimulate transactiviation of one or all steroid receptors, (4) the ability to reverse ER squelching of hPR activation in a dose dependent manner; and/or (5) the ability of a truncated form of the SRC-1 polypeptide to inhibit receptor activity in a dominant negative manner. The amino acid sequence is preferably substantially similar to the sequence shown in FIGS. 1A-1E, or fragments thereof. A sequence that is substantially similar will have at least 90% identity (preferably at least 95% and most preferably 99-100%) to the sequence of FIGS. 1A-1E.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid is meant a polymer of 2 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. In certain embodiments of the invention longer nucleic acids are preferred, for example those of 1202, 1221, 1239, 1275 or more nucleotides and/or those having at least 50%, 60%, 75%, 90%, 95% or 99% identity to the full length sequence shown in FIGS. 1A-1E. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it and thus is meant to distinguish from isolated chromosomes.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
In preferred embodiments the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in the full length amino acid sequence of FIGS. 1A-1E, a functional derivative thereof, or encodes at least 75, 90, 105, 120, 150, 475, 490, 505, 520, or 550 contiguous amino acids thereof; the SRC-1 polypeptide comprises, consists essentially of, or consists of at least 25, 30, 35, or 40 contiguous amino acids of a SRC-1 polypeptide. The nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be mammalian (human) blood, semen, or tissue and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer. In yet other preferred embodiments the nucleic acid is a conserved or unique region, for example those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, and obtaining antibodies to polypeptide regions.
By xe2x80x9cconserved nucleic acid regionsxe2x80x9d, are meant regions present on two or more nucleic acids encoding a SRC-1 polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding SRC-1 polypeptides are provided in Abe, et al. J. Biol. Chem., 19:13361 (1992) (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 5 out of 20 nucleotides.
By xe2x80x9cunique nucleic acid regionxe2x80x9d is meant a sequence present in a full length nucleic acid coding for a SRC-1 polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably comprise 30 or 45 contiguous nucleotides present in the full length nucleic acid encoding a SRC-1 polypeptide. In particular, a unique nucleic acid region is preferably of mammalian origin.
The invention also features a nucleic acid probe for the detection of a SRC-1 polypeptide or nucleic acid encoding a SRC-1 polypeptide in a sample. The nucleic acid probe contains nucleic acid that will hybridize to a sequence set forth in FIGS. 1A-1E or a functional derivative thereof. The SRC-1 polypeptide that is detected may comprise, consist of, or consist essentially of any given number of contiguous amino acids of the amino acid sequence set forth in FIGS. 1A-1E.
By xe2x80x9ccomprisingxe2x80x9d it is meant including, but not limited to, whatever follows the word xe2x80x9ccomprisingxe2x80x9d. Thus, use of the term xe2x80x9ccomprisingxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting ofxe2x80x9d is meant including, and limited to, whatever follows the phrase xe2x80x9cconsisting ofxe2x80x9d. Thus, the phrase xe2x80x9cconsisting ofxe2x80x9d indicates that the listed elements are required or mandatory, and that no other elements may be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase xe2x80x9cconsisting essentially ofxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
In preferred embodiments the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 25, 50, 75, 90, 100, 120, 150, 412, 425, 450, 475, 490, 500, 520, or 550 contiguous amino acids of the full-length sequence set forth in FIGS. 1A-1E or a functional derivative thereof. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Under stringent hybridization conditions only highly complementary, nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.
Methods for using the probes include detecting the presence or amount SRC-1 RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to SRC-1 RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a SRC-1 polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed., 1992) hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
The invention also features recombinant nucleic acid, preferably in a cell or an organism. The invention also provides a recombinant cell or tissue containing a purified nucleic acid coding for a SRC-1 polypeptide. The recombinant nucleic acid may contain a sequence set forth in FIGS. 1A-1E or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complimentary to an RNA sequence encoding a SRC-1 polypeptide and a transcriptional termination region functional in a cell. In such cells, the nucleic acid may be under the control of its genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By xe2x80x9cexogenousxe2x80x9d it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the SRC-1 polypeptide.
In other aspects, the invention provides transgenic, nonhuman mammals containing a transgene encoding a SRC-1 polypeptide or a gene effecting the expression of a SRC-1 polypeptide. Such transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introducing a SRC-1 polypeptide, regulating the expression of a SRC-1 polypeptide (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes).
A xe2x80x9ctransgenic animalxe2x80x9d is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. The transgenic DNA may encode for a human SRC-1 polypeptide. Native expression in an animal may be reduced by providing an amount of anti-sense RNA or DNA effective to reduce expression of the receptor.
In other embodiments a steroid ligand activates a molecular switch (as described herein and in is U.S. patent application Ser. No. 07/939,246, filed Sep. 2, 1992 and 08/479,913, filed Jun. 7, 1995 both of which are incorporated herein by reference in their entirety including any drawings) and the SRC-1 polypeptide and thereby provides a super-physiological response to enhance steroid therapy. Expression of the SRC-1 polypeptide may be driven by a constitutively active promoter with a coding region for SRC-1. The gene switch may optionally be provided either in the same plasmid or in a different plasmid. The promoter for SRC-1 may be regulated by a gene switch so that the ligand activates both SRC-1 and the gene of interest.
In another aspect the present invention provides a method for increasing the transcription of a target gene. Transcription refers to the process of converting genetic information from DNA to RNA. The method involves the step of providing nucleic acid encoding a SRC-1 polypeptide to a cell containing said target gene. The increase may be from an initial level of no transcription or may be from a pre-existing level of transcription. The target gene can be any gene that is transactivated by SRC-1. The level of transcription may be determined using methods known in the art; for example the level of transcription may be assessed by measuring the chloramphenicol acetyl transferase activity. Providing SRC-1 nucleic acid, or the polypeptide itself, to a cell can increase the transcriptional activity of any steroid receptor, such as the mineral corticoid (MR), androgen (AR), estrogen, progesterone, Vitamin D, COUP-TF, cis-retonic acid, Nurr-1, thyroid hormone, mineralocorticoid, glucocorticoid-xcex1, glucocorticoid-xcex2 and orphan receptors.
In preferred embodiments the method may also involve the step of providing a molecular switch for regulating expression of a nucleic acid cassette in gene therapy to the cell containing the target gene. The molecular switch includes a natural steroid receptor DNA binding domain linked to a modified ligand binding domain. Preferably the SRC-1 polypeptide comprises the full length amino acid sequence of FIGS. 1A-1E (SEQ ID NO:5)or a fragment thereof having at least 700, 800, or 900 contiguous amino acids of the full length sequence, or a fragment containing an essential interaction domain of SRC-1. The switch is preferably tissue specific, as described herein.
The method may also involve: (1) attaching the molecular switch to a nucleic acid cassette to form a nucleic acid cassette/molecular switch complex for use in the gene therapy; (2) administering a pharmacological dose of the nucleic acid cassette/molecular switch complex to an animal or human to be treated; (3) turning the molecular switch on or off by dosing the animal or human with a pharmacological dose of a ligand which binds to the modified ligand binding site; and (4) transcribing the nucleic acid to produce a protein after the animal or human is given a pharmacological dose of the ligand. These steps are described and definitions for terms such as xe2x80x9cnucleic acid cassettexe2x80x9d, and xe2x80x9cplasmidxe2x80x9d are provided in U.S. patent application Ser. No. 07/939,246, filed Sep. 2, 1992 and International Patent Publication WO 93/23431, published Nov. 25, 1993, both of which are incorporated herein by reference in their entirety including any drawings.
The molecular switch and the nucleic acid cassette may be on the same or separate plasmids and may be co-injected into a target cell or injected separately. Similarly, the molecular switch and the nucleic acid encoding the SRC-1 polypeptide may be on the same or separate plasmids and may be co-injected or separately injected into a target cell.
In another aspect the invention provides a composition of matter comprising a molecular switch linked to a nucleic acid cassette. The cassette/molecular switch complex is positionally and sequentially oriented in a vector such that the nucleic acid in the cassette can be transcribed and when necessary translated in a target cell. The molecular switch regulates a constitutively active promoter in a plasmid with a coding region for a SRC-1 polypeptide.
The invention also features a method for decreasing the transcription of a target gene. The method involves providing nucleic acid encoding a dominant-negative inhibitor of a SRC-1 polypeptide in a cell containing said target gene. The dominant negative inhibitor preferably is encoded by a N truncated fragment of the full length sequence, such as the approximately 150 amino acid long fragment of Example 8.
In another aspect the present invention provides a molecular switch for regulating expression of a nucleic acid cassette in gene therapy, comprising a modified SRC-1 polypeptide, said polypeptide including a natural SRC-1 activation domain linked to a modified binding domain. In this embodiment the SRC-1 nucleic acid forms part of the molecular switch. Thus, a substitution is envisioned in a previosly described switch which included a DNA binding domain of a steroid receptor (for example GAL-4) linked to a transactivation domain (for example VP-16) linked to ligand binding domain (for example a mutated LBD of the progesterone receptor). The substitution involves replacing VP-16 or some other transactivation domain with an essential interaction domain of SRC-1. The term xe2x80x9cessential interaction domainxe2x80x9d refers to the portion of SRC-1 required for interaction with other transcriptional factors and agents and those skilled in the art may locate an essential interaction domain using techniques known in the art.
The invention also features a method for regulating expression of a nucleic acid cassette in gene therapy comprising the step of attaching a modified SRC-1 polypeptide molecular switch to a nucleic acid cassette to form a nucleic acid/molecular switch complex for use in gene therapy and administering a pharmacological dose of the nucleic acid cassette/molecular switch complex to an animal or human to be treated.
In another aspect the invention features a composition of matter comprising a modified SRC-1 polypeptide molecular switch linked to a nucleic acid cassette, wherein said complex is positionally oriented in a vector such that the nucleic acid in the cassette can be transcribed and when necessary translated in a target cell.
The invention also features a method of treating a SRC-1 related disease or condition (such as those described herein which require modulation of steroid receptor activity) comprising the steps of inserting an expression vector containing a SRC-1 coding sequence into cells, growing the cells in vitro, and infusing the cells into a patient in need of such treatment.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.